Mutations of myosin XVA in humans and mice cause profound congenital deafness. Myosins are actin based molecular motors that have a conserved head (motor) and neck (light chain binding motifs) and highly divergent tail domains. In collaboration with Dr. Jim Sellers and Dr. Fei Wang at the National Heart Lung and Blood Institute, we are using a baculovirus expression system to purify MYO15A protein consisting of the head and neck domains. The recombinant proteins will be used to study the actin binding, ATP hydrolysis and motility properties of MYO15A. The in vitro motor domain assay will provide us with a means of examining the effect of these mutations on myosin XV motor function. The MYO15A tail contains several domains which are candidates for protein interaction motifs. The identification of proteins that functionally interact with MYO15 may provide the best means of determining the role of MYO15A in the auditory system. In addition, interacting proteins are themselves likely to play crucial roles in hearing and would be strong candidates for proteins encoded by deafness loci. We are therefore using the yeast two hybrid system and phage display to identify proteins that interact with the myosin XVA. Genes that encode poteins that interact with myosin XVA from these two screens will be further examined for biological relevance. In addition, using confocal microscopy, we are transfecting wild type and mutated forms of GFP-Myo15a and other unconventional myosins contructs into organ of Corti explant cultures in order to study in vivo targeting and tethering of mysoin XVa at the tips of stereocilia. recently we demonstrated that whirlin is cargo of myosin XVa and is transported to the tips of stereocilia by myosin XVA. Both of these proteins are necessary for elonagtion and staircase formation of the stereocilia bundle (Belyantseva et al. 2005).